A β-hairpin structure for self-assembly to capture antimicrobial peptide JR-RF and its application in the preparation of antimicrobial agents.

Abstract

This invention discloses a β-hairpin structure for self-assembling and capturing an antimicrobial peptide J-R-RF and its application in the preparation of antimicrobial agents, relating to the field of biomedical technology. The amino acid sequence of this β-hairpin structure for self-assembling and capturing an antimicrobial peptide J-R-RF is shown in SEQ ID NO. 6. This invention selects a broad-spectrum antimicrobial short peptide J-1 derived from bee royal jelly as the template peptide. Based on the amino acid sequence characteristics of short peptide J-1, it designs and modifies the structure to obtain alternating hydrophilic and hydrophobic amino acid structural units, which are then used as the backbone of the self-assembled peptide fiber. D The β-hairpin peptide is designed using two turning sequences, PG and RRRF, to achieve a dual antibacterial and bacterial trapping effect. The β-hairpin structure self-assembled to trap antimicrobial peptides provided by this invention can form a fibrous network structure, exhibiting good antibacterial effect and high stability, as well as excellent biocompatibility, providing effective technical support for the development of novel antimicrobial drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a β-hairpin structure for self-assembling and capturing antimicrobial peptide JR-RF and its application in the preparation of antimicrobial agents. Background Technology

[0002] Antimicrobial peptides, as an important component of the innate immune system, exert their antibacterial effects through physical mechanisms such as altering membrane permeability and disrupting cell membrane integrity, and are considered promising alternatives to traditional antibiotics. However, natural AMPs suffer from a series of problems, including weak biological activity, high toxicity, and immature stability, which seriously hinder their application. In recent years, advances in molecular design and optimization strategies, as well as the rapid rise of nanotechnology, have shown the potential to improve the biological and chemical properties of AMPs, making their application possible.

[0003] Hydrogen bonds, ionic interactions, hydrophobic interactions, and π-π stacking interactions between amino acids are the main driving forces for self-assembly. Supramolecular self-assembly is considered one of the methods to improve the antibacterial activity and stability of antimicrobial peptides. This invention aims to develop nanostructured antimicrobial peptides with self-assembly tendency, in order to provide technical support for avoiding antibiotic resistance and developing effective clinical anti-infective alternatives. Summary of the Invention

[0004] The purpose of this invention is to provide a β-hairpin structure self-assembled trapping antimicrobial peptide JR-RF and its application in the preparation of antimicrobial agents, thereby solving the problems existing in the prior art. The β-hairpin structure self-assembled trapping antimicrobial peptide JR-RF provided by this invention has good antibacterial effect and high stability, and excellent biocompatibility, providing effective technical support for the development of novel antimicrobial drugs.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a β-hairpin structure for self-assembly to capture an antimicrobial peptide JR-RF, the amino acid sequence of which is shown in SEQ ID NO. 6.

[0007] The present invention also provides the application of the above-mentioned β-hairpin structure self-assembly captured antimicrobial peptide JR-RF in the preparation of antimicrobial agents, wherein the antimicrobial agents target pathogens such as Escherichia coli, Cronobacter sakazakii, Salmonella typhimurium, Pseudomonas aeruginosa, Salmonella, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus albus, Mycobacterium abscessum, or Bacillus cereus.

[0008] The present invention also provides an antibacterial agent, the active ingredient of which includes the above-mentioned β-hairpin structure self-assembled trapping antibacterial peptide JR-RF.

[0009] The present invention discloses the following technical effects:

[0010] This invention selects Jelleine-1 (J-1), a broad-spectrum antibacterial short peptide derived from bee royal jelly, as the template peptide. Based on the amino acid sequence characteristics of short peptide J-1, it designs and modifies the structure to obtain alternating hydrophilic and hydrophobic amino acid structural units, which are then used as the self-assembled peptide fiber backbone. D The β-hairpin peptide, designed using two turning sequences, PG and RRRF, exhibits dual antibacterial and bacterial trapping effects. The self-assembled trapping antimicrobial peptide in this invention can form a fibrous network structure, demonstrating excellent antibacterial efficacy, high stability, and superior biocompatibility, providing effective technical support for the development of novel antimicrobial drugs. Attached Figure Description

[0011] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0012] Figure 1 High-performance liquid chromatograms of template peptide J-1 and designed peptides J-1-pG and J-1-RF;

[0013] Figure 2 To design high-performance liquid chromatograms for peptides JR, JR-pG, and JR-RF;

[0014] Figure 3 Mass spectra of template peptide J-1 and designed peptides J-1-pG and J-1-RF;

[0015] Figure 4 Mass spectra of peptides JR, JR-pG, and JR-RF were designed.

[0016] Figure 5 Circular dichroisms of template peptide J-1 and designed peptides J-1-pG, J-1-RF, JR, JR-pG and JR-RF in three different environments;

[0017] Figure 6 A statistical graph showing the toxicity of template peptide J-1 and designed peptides J-1-pG, J-1-RF, JR, JR-pG and JR-RF to RAW264.7 macrophages;

[0018] Figure 7The results are for the determination of the critical aggregation concentration of β-hairpin; where A is the fluorescence intensity of hairpin J-1-RF at different concentrations; B is the linear fitting graph of the concentration of hairpin J-1-RF and fluorescence intensity; C is the fluorescence intensity of hairpin JR-pG at different concentrations; and D is the linear fitting graph of the concentration of hairpin JR-pG and fluorescence intensity.

[0019] Figure 8 Transmission electron microscope images of β-hairpin peptide J-1-RF at different concentrations;

[0020] Figure 9 Atomic force microscopy for β-hairpin J-1-RF;

[0021] Figure 10 The results of the β-hair peptide bacterial capture ability assay are shown in Figure A; where A is the cuvette observation chart and B is the statistical chart of CFU values ​​of Escherichia coli. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0026] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0027] Example 1: Peptide Sequence Design and Physicochemical Parameter Analysis

[0028] The invention selects Jelleine-1 (i.e., J-1), a broad-spectrum antimicrobial peptide derived from royal jelly, as a template. The first amino acid at its N-terminus is replaced with arginine, and a β-sheet structure with alternating hydrophilic and hydrophobic amino acids is constructed as the β-hairpin structure self-assembled peptide fiber backbone. D Sequence design was performed using two turning sequences, PG and RRRF, resulting in four β-hairpin peptides (J-1-pG, J-1-RF, JR-pG, and JR-RF). The physicochemical parameters and molecular structures of all tested peptides are shown in Table 1 and [Table data missing]. Figure 1 As shown.

[0029] Table 1. Amino acid sequence and physicochemical parameters of the target peptide

[0030]

[0031] Note: a) Actual molecular mass was determined by mass spectrometry; b) Isoelectric point was calculated using the online tool PepCalc.com - Peptidecalculator; p represents D P stands for D-proline.

[0032] Hydrophilic cationic residues and hydrophobic residues play important roles in electrostatic interactions with negatively charged bacterial membranes and in facilitating peptide sequence penetration into hydrophobic domains of the bacterial membrane, respectively. In this study, the designed peptides exhibited increased positive charges compared to the template peptides, providing appropriate electrostatic interactions for antibacterial activity.

[0033] Example 2 Synthesis and Identification of Polypeptides

[0034] 1. Synthesis of antimicrobial peptides

[0035] The polypeptides shown in Table 1 were synthesized using a solid-phase synthesis method:

[0036] Weigh the resin, soak it in dichloromethane for 5 min, wash with N,N-dimethylformamide, remove the dichloromethane, and then remove the Fmoc protecting group on the resin with the prepared deprotecting agent (decapping) for 20 min. Weigh the second amino acid at the C-terminus, add the condensing agent N,N-dimethylformamide, and place it in the reaction column for reaction (gas blowing). Detect the result using the ninhydrin method; the solution is bright yellow, the resin is transparent, and there are no impurities. Remove the Fmoc protecting group on the second amino acid with the deprotecting solution, wash 6 times with N,N-dimethylformamide, and repeat the operation until the last amino acid. Remove the Fmoc protecting group on the last amino acid with the deprotecting solution, and then wash with N,N-dimethylformamide. After the reaction, wash with dichloromethane and methanol respectively to shrink the peptide. Digest the peptide with lysis buffer and precipitate the liquid with diethyl ether. Analyze the purified peptide using mass spectrometry (MS) and high-performance liquid chromatography (HPLC).

[0037] 2. Purification of antimicrobial peptides

[0038] (1) Pre-analysis: Take the sample and put it into a 0.5 mL tube and dissolve it with ultrapure water. Filter the sample through a 0.45 μm membrane, and then analyze the sample injection using rapid gradient high performance liquid chromatography (10-100%).

[0039] (2) Sample preparation: Add 200 mg of sample to a 20 mL beaker, then add 15 mL of H2O and 5 mL of methanol. Sonicate the sample until it is completely dissolved, and then filter the solution through a 0.45 μm membrane.

[0040] (3) High-performance liquid chromatography (HPLC): Collect samples from 0 to 25 min. Pump A contains 100% acetonitrile plus 0.1% trifluoroacetic acid, and pump B contains 100% water plus 0.1% trifluoroacetic acid. Analyze the collected components using HPLC to check their purity.

[0041] Chromatograms of the template peptide and the designed peptide after purification are shown below. Figures 1-2 As shown in the figure, the results indicate that each designed peptide has a distinct absorption peak. The purity of the purified peptides reached over 95%.

[0042] 3. Identification of antimicrobial peptides

[0043] The purified peptides were collected and the target peaks were identified using an LC 6000 reversed-phase preparative chromatograph and a Waters 2000 mass spectrometer. Finally, the actual peptide molecular weight and purity were analyzed. The mass spectrometric analysis of the purified peptides is as follows: Figures 3-4As shown in the figure. The results showed that the actual relative molecular mass was basically consistent with the theoretical value, indicating that all peptides were successfully synthesized.

[0044] Example 3 Determination of the secondary structure of antimicrobial peptides

[0045] 1. Determination of the secondary structure of antimicrobial peptides

[0046] The secondary structures of the antimicrobial peptides (shown in Table 1) were determined by circular dichroism spectroscopy (CD).

[0047] Structures were constructed in three simulated solution environments (aqueous environment, prokaryotic cell membrane environment, and hydrophobic environment of biological membrane). The peptides were dissolved in 10 mM PBS buffer (pH 7.4), 30 mM sodium dodecyl sulfate (SDS), and 50% trifluoroethanol (TFE) to prepare peptide solutions with a final concentration of 150 μM. Circular dichroism spectroscopy was used to scan the peptides in the wavelength range of 190 nm–250 nm at room temperature, with a scan rate of 1 nm / s. Results are shown below. Figure 5 As shown.

[0048] In PBS buffer, J-1 exhibits a negative absorption peak at 198 nm, displaying a random coil conformation. However, in SDS buffer, JR, J-1-pG, JR-pG, and JR-RF show positive absorption peaks at 203 nm and negative absorption peaks around 216 nm, indicating typical β-sheet structures. This suggests that the alternating arrangement of hydrophilic and hydrophobic amino acids facilitates the formation of a β-sheet conformation of the peptide in a simulated hydrophobic environment.

[0049] Example 4: Determination of antibacterial activity

[0050] Determination of minimum inhibitory concentration (MIC): Antimicrobial activity was determined using the micro-dilution method. The test strains were cultured to the logarithmic growth phase, and different concentrations of peptide (as shown in Table 1) and bacterial culture were added to rows 1-11 of 96-well plates, respectively, and incubated at 37°C for 16 h. Bacterial culture without peptide and culture medium were used as positive and negative controls, respectively. The MIC value of the peptide is expressed as the critical concentration at which no visible turbidity or precipitation occurs. The results are shown in Tables 2 and 3.

[0051] Table 2. MIC values ​​(μM) of short peptides against Gram-negative bacteria.

[0052]

[0053] Table 3. MIC (μM) of short peptides against Gram-positive bacteria.

[0054]

[0055] All five derived peptides (JR, J-1-pG, J-1-RF, JR-pG, and JR-RF) exhibited broad-spectrum antibacterial activity, and their antibacterial activity was significantly enhanced compared with that of the template peptide. Among them, the designed peptides JR and J-1-RF showed enhanced antibacterial activity, which may be attributed to the appropriate increase in the number of positive charges.

[0056] Example 5 Cytotoxicity Detection

[0057] The cytotoxicity of peptides J-1, JR, J-1-pG, J-1-RF, JR-pG, and JR-RF was detected.

[0058] The cytotoxicity of peptide-treated mouse macrophages (RAW 264.7) was evaluated using a CCK-8 assay kit. In summary, cells were seeded into individual wells of 96-well plates and exposed to different concentrations of peptide solutions at 37°C and 5% CO2 for 12 h. After incubation, 10 μL of CCK-8 solution was added to each well, and the cells were incubated for 2 h. The absorbance at 450 nm was then measured using a microplate reader. Positive controls consisted of untreated cells, and negative controls consisted of intact culture medium.

[0059] like Figure 6 As shown, experiments revealed that cell viability gradually decreased with increasing concentration; at a concentration of 4 μM, the cell viability of bee venom was below 20%. In contrast, the peptides designed in this invention maintained a cell viability greater than 80% at a test concentration of 64 μM. These results demonstrate that the series of derived peptides designed in this invention exhibit good biocompatibility.

[0060] Example 6: Determination of Critical Aggregation Concentration (CAC)

[0061] The CAC of peptides J-1, JR, J-1-pG, J-1-RF, JR-pG, and JR-RF was determined using the ANS fluorescence assay.

[0062] ANS was dissolved in dimethylformamide at a concentration of 1 mM, and then 1 μL of this solution was added to 100 μL of peptides of different concentrations. The mixtures were transferred to quartz cuvettes and measured using an F-4500 fluorescence spectrophotometer (Hitachi, Japan), collecting fluorescence spectra from 420 to 550 nm under 360 nm excitation. CAC was determined by matching curves, obtained by plotting the ANS fluorescence intensity at 475 nm for different peptide concentrations. Results are as follows: Figure 7 As shown.

[0063] The results showed that the critical aggregation concentrations of the two hairpin peptides, J-1-RF and JR-pG, were 12.45 μM and 13.03 μM, respectively. Therefore, above these critical concentrations, the hairpin peptides can self-assemble into nanostructures, thereby achieving excellent bioactivity and stability.

[0064] Example 7 Characterization of self-assembled trap peptide fiber structure

[0065] To observe the formation of the self-assembled structure of β-hairpin peptides, the microstructure of the peptides was observed using transmission electron microscopy with negative staining and atomic force microscopy to assess their ability to form fibrillary structures. The steps are as follows:

[0066] 10 μL of different concentrations of hairpin peptide J-1-RF solution were evenly spread onto a copper grid (400 square mesh), allowed to adsorb for 5 min, stained with 0.1% phosphotungstic acid for 30 s, dried at room temperature for 15 min, and observed using a transmission electron microscope. The results are as follows: Figure 8 As shown.

[0067] The results showed that when the J-1-RF concentration was 12 μM, the nanofibers observed by transmission electron microscopy were only short and thin, indicating that they were in the early stage of aggregation. When the concentration was increased to 32 μM and 128 μM, the hairpin peptide J-1-RF further self-assembled into supramolecular nanostructures. The antiparallel β-sheet dominated and drove the formation of twisted and dense fiber structures, indicating concentration-driven self-assembly. Atomic force microscopy was used to investigate the formation of fiber morphology by J-1-RF hairpin peptide (32 μM), and the same results were obtained. Figure 9 ).

[0068] Example 8 Bacterial Agglutination Test

[0069] To evaluate the ability of antimicrobial peptides to capture pathogens, peptides J-1, JR, J-1-RF, and JR-pG were mixed with Escherichia coli bacterial suspensions in sterile cuvettes. Bacterial aggregation was observed, and the supernatant of the bacterial suspensions was collected, serially diluted, and viable bacteria were counted.

[0070] The specific experimental steps are as follows:

[0071] (1) Cell preparation: Escherichia coli ATCC 25922 was used as the model strain to obtain bacterial culture that had grown to the logarithmic phase. The culture was centrifuged at 3000 rpm for 5 min and washed 3 times with HEPES buffer (pH=7.0).

[0072] (2) Co-culture of peptides and bacteria: Take OD 600 3 mL of bacterial suspension with a concentration of 0.25 was added to a cuvette sterilized with ethanol, followed by 32 μM nanopeptide solution. The mixture was left to stand at room temperature for 8 hours. The bacterial aggregation state was observed and photographed. The untreated group was used as the control group.

[0073] (3) Result evaluation: At 0, 30, 60 and 90 min, 50 μL of bacterial supernatant was taken and serially diluted in 450 μL of autoclaved PBS. 100 μL of the diluted solution was then inoculated evenly onto MHA plates. The plates were incubated overnight at 37°C. The number of colonies in each sample was counted, and the CFU value of each sample was calculated. The experiment was repeated three times.

[0074] The results are as follows Figure 10 As shown, the results revealed that with increasing time, β-hair peptide induced rapid aggregation and sedimentation of Escherichia coli, and the total number of colonies in the supernatant decreased significantly. Among them, J-1-RF could induce complete sedimentation of bacteria after 90 min, demonstrating a strong bacterial capture effect.

[0075] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A β-hairpin structure for self-assembling and capturing the antimicrobial peptide JR-RF, characterized in that, The amino acid sequence is shown in SEQ ID NO.

6.

2. The application of the β-hairpin structure self-assembly capturing antimicrobial peptide JR-RF as described in claim 1 in the preparation of antimicrobial agents, characterized in that, The antibacterial agent targets pathogens such as Escherichia coli, Cronobacter sakazakii, Salmonella typhimurium, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus albus, Mycobacterium abscessum, or Bacillus cereus.

3. An antibacterial agent, characterized in that, The active ingredient includes the β-hairpin structure self-assembly captured antimicrobial peptide JR-RF as described in claim 1.

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